The electrospray ionization process and add-on device with sample injection tip relate generally to electrospray ionization (ESI) processes and an add-device having an ESI sample injection tip and additional components for performing various sample preparation processes, such as, for example, gel electroelution and sample filtering/separating. More particularly, the electrospray ionization process relates to an electrospray ionization process having as much as 50 percent improvement in signal, and the add-on device can have a sample injection tip for such ESI process. Complimentary sample preparation devices can also be provided including a gel electroelution device and a separator device with tangential fluid flow.
Basically, “electrospray” is a method of generating a very fine liquid aerosol through electrostatic charging. In electrospray, a liquid is passed through a nozzle, and an electric potential is applied to the liquid either externally through conductive fittings or internally in-line with the solution flow path. There exists an air gap between the nozzle and the electrical ground whose resistance is large, and thus requires a large electrical potential relative to ground. When the applied voltage is of sufficient magnitude, the liquid solution can be induced to bridge the air gap and complete the electrical circuit. Essentially, as the applied voltage is increased, charge density builds at the tip of the nozzle and increases proportionally to the potential until the point at which the electric potential pull on the ions is greater than the reverse force of surface tension on the drop. As the charge density increases, fine droplets of liquid break away from the nozzle tip and form fine aerosol droplets (referred to as the plume) that transit the air gap to the electrical ground. During transit, the fine aerosol droplets are dried via passive and/or active evaporation, resulting in decreased droplet size and a consequent increase in charge density within the droplets. After a continued reduction in overall size, the increased Coulombic repulsions within the diminishing droplets lead to ejection of ions into the gas phase.
When used as a soft ionization method for chemical analysis, it is commonly called “electrospray ionization,” or ESI. Here, ionization is the process of generating a gas phase ion from a liquid (typically) or solid chemical species. In this context, the method is characterized as “soft” because the chemical species are ionized by manipulating the solvent properties (i.e. pH), and not through collisional processes, as in “hard” ionization. Ionization is a critical event in mass spectrometry because the instrument functionally determines a molecule's mass-to-charge ratio, which requires that the molecule has a charge to be analyzed.
Electrospray ionization processes are well known in the art. A review of the historical development of electrospray ionization can be found in U.S. Pat. No. 6,462,337, by Li, et al. Electrospray ionization is commonly utilized in the analysis of biological samples, such as proteins and peptides that are susceptible to thermal degradation. Ionization of sample molecules can be controlled by adjusting the pH of the solvent to manipulate protonation and deprotonation events.
In mass spectrometric analysis, the basis for ESI is the establishment of an electrical circuit between the mass spectrometer and an electrode from the mass spectrometer in the flow path of the sample solution. A small air gap bridges the distance between the sample injection tip (i.e. tip emitter) and the mass spectrometer. When a voltage (1 to 5 kV) is applied to the liquid sample flowing through the ESI tip, a region of high charge density is produced at the outlet of the tip emitter. The electrospray process occurs at the outlet of the tip emitter. As the liquid begins to exit the tip emitter, it charges up and assumes a conical shape, referred to as a Taylor cone, so named after G. I. Taylor who described the phenomena in 1964. In general, the Taylor cone is formed due to the competing forces of the static electric field and the liquid's surface tension. The liquid assumes a conical shape because the force of the electric field can be felt at the apex of the cone as an extension of Newton's first law, which is commonly known as “the path of least resistance”. Thus, the liquid, which is malleable, will transform to react to the potential to the extent that the force of the surface tension supplies an equal resistance. Therefore, at the tip of the Taylor cone, the liquid changes shape into a fine jet. However, this jet becomes unstable, and breaks up into a mist of fine, highly charged droplets, called the “plume.” Desolvation of the droplets in the plume, combined with coulombic repulsion, leads to the production of gas phase ions. The continuity of the electrical circuit is completed as the ions transit the air space to the mass spectrometer. As soon as the ions enter the mass spectrometer, they are directed to the ion trap by ion optics for subsequent detection and determination of their mass-to-charge ratio. Thus, the ESI process is initiated by creating a Taylor cone of the fluid sample at the terminus of the aperture/sample injection tip, which can be referred to interchangeably as an “ESI tip,” “ESI tip emitter,” “tip emitter,” or simply “emitter.”
A conventional electrospray device for mass spectrometry can comprise, for example, an ESI tip in fluid communication with a sample liquid to be analyzed, and associated with a high voltage power supply. The ESI tip is positioned so as to emit the ionized liquid sample into a mass spectrometer. The ESI tip can be, for example, a hollow metal tube, e.g., a syringe needle. The liquid sample is passed through the tip, for example hydrodynamically, and the high-voltage power supply can be connected to the outside of the ESI tip, especially if the tip is electrically conductive or has a conductive coating. If the ESI tip is made from a non-conductive material, and the outside was not coated with a conductive material, the voltage could be applied to the liquid. The ESI tip is positioned in front of a plate, called a counter-electrode, which is commonly held at ground potential. When the power supply is turned on and adjusted for the proper voltage, the liquid sample flowing through the ESI tip transforms into the aforesaid Taylor cone as it is emitted from the ESI tip, and then ultimately ionizes into a fine continuous mist of highly charged droplets that transit the air space toward the counter-electrode.
Many different types of ESI tips are known in the art for use in combination with a mass spectrometer. All known conventional ESI tips are designed to have the “sharpest” tip possible. Conventional tip emitters can have an aperture diameter (ID) of around 25 μm or less, and an outer diameter (OD) as small as possible, i.e., the tip is made as “sharp” as possible. An example of a recently developed commercially available ESI tip is a fused silica capillary tip. This tip is formed by a process in which the capillary is heated and “pulled” to a fine pointed tip. Devices such as the Sutton Instrument laser puller can use laser light to heat the fused silica capillary while providing a pulling tension that draws the capillary into a much smaller diameter. When the stress point at the constriction becomes too great for the capillary, the constriction breaks into two sharp, i.e., finely pointed, tips. New Objective, Inc., of Woburn, Mass. produces fused silica capillary ESI tips in this fashion. Conductive coatings, such as gold, silver and polyaniline, can be placed on these ESI tips to permit direct connection of the electrode to the tip's exterior surface. Conventional stainless steel tip emitters are also commercially available from vendors such as from New Objective and Thermo Finnigan, Ltd., Hertfordshire, UK. Small hollow stainless steel tubes can either be fabricated with a small constant inner diameter, or drawn down to fine tip points on one end. These tips are naturally conductive, and the electrospray voltage can be directly applied to the outer surface of the tip. Advion Biosciences, Ithaca, N.Y., has also developed a silicon tip emitter for “static” ESI mass spectrometry. In a static ESI process, fluid flow through the column is not controlled by hydrodynamic pumping. Instead, a small amount of sample solution is injected into an inlet end of the tip, and capillary action draws the sample solution towards the outlet end of the tip at very low flow rates.
All “ESI tips” must create a Taylor cone in order to properly introduce the sample ions into the gas phase, where they can be steered by the electrical ion optics controlled within the mass spectrometer. Without this charge, the ions would pass straight to the vacuum waste and not be detected. Essentially, surface tension prevents the very tiniest drops of liquid from breaking free. If the drops are too large, the excess solvent causes formation of hydrates and other adjuncts, which interferes both with electrical steering of the ions in the ion optics and with accurate determination of the ion's mass-to-charge ratio, as the instrument will detect the combined mass of the complexes To overcome surface tension, organic solvent can be added. To break free, a high electric field (e.g., 2,000 V) can be applied to the liquid/tip, with the mass spectrometer being grounded. The high voltage is what breaks the micro droplets free from the bulk liquid. One way to concentrate the electric field (V/cm2) without increasing the voltage is to minimize the area. This method is likely the reason that all (known) prior art, and current literature, teaches shrinking the ESI tip diameter to as small/sharp a point as possible.
Heretofore, it has been atypical to combine mass spectrometer sample preparation and sample injection in a single system or device. In part this is attributable to the very different scientific traditions of sample preparation (generally wet chemistry of some kind) versus the traditional biophysics of mass spectrometer sample injection and analysis. However, the prior art combination of sample preparation and injection is not unknown. For example, U.S. Pat. No. 6,942,793 discloses a liquid chromatography mass spectrometer in which a number of devices are combined in a single system. Known systems can include a pump, sample injector, plurality of separation columns including a first separation column and a second separation column, and a mass spectrometer. Such systems can also include a plurality of trap columns for retaining a sample component separated by the first separation column and a first switching valve for switching among one of the plurality of trap columns and another one of the plurality of trap columns at regular time intervals in such a way that when one of the plurality of trap columns is connected to the first separation column, another one of the plurality of trap columns is connected to the pump, and vice versa. A second switching valve can be employed to enable a trap column that is connected to the pump to be further connected to the second separation column, with the second separation column being connected to the mass spectrometer and capable of separating the sample component in a shorter time than the first separation column. Overall, U.S. Pat. No. 6,942,793 identifies the general benefits of combining sample preparation and injection in an overall coordinated system—a system to which the present invention nonetheless provides significant additional advantages as explained herein.
Continued challenges in sample preparation for mass spectrometry injection have to do with either or both of sample composition and/or contamination. For example, contamination in samples of interest is a serious problem in mass spectrometry. Mass spectrometry samples are so small that, literally, the wave of an ungloved hand near an exposed sample, or sample precursor, can deposit, sight unseen, enough keratin or other proteins from shed skin to skew the composition significantly. Also, preparation of mass spectrometry samples of biological materials usually requires the removal of abundant proteins, such as the ubiquitous albumin, to enrich the relative concentrations of the peptides or proteins of interest, and such preparation in turn needs to be conducted in a way that is both fast and efficient. Interchangeability is an issue, too. Just as in past decades users had to schedule and share their use of mainframe computers, today, mass spectrometry personnel need easy, efficient and contamination-free ways to share a single (very expensive) mass spectrometer.
Known ESI processes have heretofore enabled significant advances in the art, however, an ESI process as described herein can provide as much as about 50 percent improvement in signal over conventional ESI process. It would also be desirable to provide an add-on device that can implement such an ESI process and that can further provide an integrated approach to mass spectrometry that enables both optimal sample preparation and avoidance of contamination, while at the same time making the mass spectrometer available to as many users as possible.